New methodology is greatly needed for hormone analysis to both keep the radioimmunoassays honest and to make up for their deficiencies regarding accuracy, sensitivity, and specificity. Recently we have achieved the direct quantitation of picogram amounts of free thyronines in human serum by a procedure involving sample clean-up, derviatization, and separation/quantitation by gas chromatography with electron capture detection (GC-ECD). We now propose to expand this methodology in two ways. First, we want to analyze total thyronines in serum and in tissues. Second, we want to alter the methodology to allow the direct analysis of peptide hormones in general. For this we propose to modify the peptide hormones with one of the three related tags, introduce the other two tags for internal standard and carrier purposes, carry out a clean-up procedure, and then quantitate the hormones based on GC-ECD of the tags. In this manner, much of the procedure will be the same for all hormones, and complete control of accuracy and specificity will be established. The proposed methodology allows some contamination to be present throughout the procedure, provides flexibility in regard to the structures of the tags and the ECD-active groups, and employs a common GC-ECD step for all hormones. The detection limit of 2 times 10 to the minus 13th power g which we have achieved on a packed column for the thyronines from actual samples, both normal and pathological, will be adequate for our work. Others (58) have reported a detection limit of 2.5 times 10 to the minus 14th power g for the ECD-active group, flophemesyl, which we plan to use for quantitation of the peptide hormones. Nevertheless, we will investigate the ability of concentration injection systems in conjunction with capillary and micropacked GC columns to extend detection limits to lower values. The final methodology is intended for reference and research purposes, and to be applicable eventually to the analysis of protein hormones.